Tools and techniques to identify, study, and control Candida auris

Candida auris, is an emerging fungal pathogen that can cause life-threatening infections in humans. Unlike many other Candida species that colonize the intestine, C. auris most efficiently colonizes the skin. Such colonization contaminates the patient’s environment and can result in rapid nosocomial transmission. In addition, this transmission can lead to outbreaks of systemic infections that have mortality rates between 40% and 60%. C. auris isolates resistant to all known classes of antifungals have been identified and as such, understanding the underlying biochemical mechanisms of how skin colonization initiates and progresses is critical to developing better therapeutic options. With this review, we briefly summarize what is known about horizontal transmission and current tools used to identify, understand, and control C. auris infections.


Introduction
Candida auris, is an emerging multidrug-resistant human fungal pathogen that predominately colonizes the skin.C. auris has been classified as an urgent threat by the US Centers for Disease Control and Prevention (CDC) Antibiotic Threats Report (2019) and ranked in the critical priority group by the World Health Organization (WHO) in a recently released list of fungal priority pathogens [1,2].C. auris is found in acute care settings and long-term care facilities and where it can spread leading to systemic infections [3][4][5][6].In some regions such as in India and parts of South Africa, C. auris has now surpassed Candida albicans as the most common Candida bloodstream species with the mortality rate ranging from 40% to 60% among infected patients [7,8].Risk factors for C. auris colonization and infection include extensive exposure to healthcare; advanced age; chronic medical conditions including diabetes mellitus, chronic renal disease, and immune compromise; and exposure to broad-spectrum antibiotics [4,9].Several isolates of C. auris have been identified that exhibit resistance to the azoles, polyenes, and echinocandins, the 3 major classes of FDA-approved antifungal drugs.The ability of C. auris to spread quickly in medical setting in coordination with this potential drug resistance poses significant challenges when attempting to treat infections [8,10,11].C. auris isolates were

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How do we identify C. auris isolates on the patient and in the environment?
C. auris infections can present similarly to other fungal infections, and drug-resistant fungal strains can phenotypically behave like antibiotic susceptible isolates when not challenged with drug.As such, it is critical the medical community have the ability to detect C. auris on the skin and quickly determine a particular culture's antifungal susceptibility.A variety of PCRbased assays have been established to identify C. auris in the healthcare environment and in waste water [24,25] and real-time PCR and loop-mediated isothermal amplification-based assays have been developed to screen for C. auris on patient's skin [26,27].In addition, standard PCR-based assays that do not require advanced infrastructure can probe for C. auris-specific GPI protein encoding genes [28].As PCRs take a couple of hours to run and process, these tests provide relatively quick surveillance of an environment or patient.In addition, T2 Magnetic Resonance system-based assays originally designed as rapid diagnostic platform to detect pathogens in blood samples have also been developed to detect C. auris 5 CFU/ml from the skin [29].
The combination of efficient environmental and patient surveillance testing is critical to the control and effective treatment of C. auris in medical facilities.One, current limitation of these assays is that drug resistance, for the most part, cannot be determined using these techniques.C. auris has been shown to use a variety of mechanisms including changes in ploidy and transcription to quickly alter drug susceptibility [30,31].As such, traditional growth assays must be used to determine antifungal susceptibility.As we learn more about C. auris drug resistance mechanisms, developing quick molecular tests such as assays to detect FKS1 mutations to determine susceptibility to echinocandins can help in the future [32].

How can we genetically manipulate C. auris?
C. auris, along with human pathogens C. albicans, C. tropicalis, and C. parapsilosis are members of the CTG fungal clade where the CTG codon is primarily translated as serine as opposed to leucine [33].C. auris displays high salt tolerance that potentially plays a role in its ability to colonize the high salt environment of the skin.In addition, isolates of C. auris have been shown to be naturally resistant to many antifungal compounds distinguishing it from other CTG species [34].Analysis of the C. auris genome sequence suggests that copy number variation and mutation of transporters, lipases, and drug targets could play roles in these phenotypes [20].The efficient and accurate manipulation of the C. auris genome is critical to understanding the underlying mechanisms that support such phenotypes.Since the C. auris was genome sequenced, a number of methodologies have been developed to determine the origins of these phenotypes [35].Forward genetic screens using Agrobacterium-mediated transformation (AtMT) have successfully been employed on C. auris [36].In AtMT, a nourseothricin (NAT r ) selectable marker is incorporated into the Agrobacterium tumefaciens Ti plasmid.This is then transformed into A. tumefaciens strain EHA105 that contains the virulence genes required for recruitment of the T-DNA.Co-culturing the modified A. tumefaciens and C. auris results in gene disruption by the plasmid while conferring nourseothricin resistance.C. auris transformants are selected based on resultant phenotypes, and whole genome sequencing is completed to identify disrupted genes.This approach has been used to screen for genes important for cellular morphology.Mutants responsible for changes in morphology were recovered in all 4 clades at rates comparable to experiments performed in other yeast species [37].Once genes of interest are identified, reverse genetics approaches can be applied to confirm the role of identified genes in the observed phenotype.
Investigators have a number of choices when applying reverse genetics to interrogate C. auris.As has been done with a variety of other fungi, direct homologous recombination methods have been employed with success to disrupt genes of interest [38].In addition, CRISPR-Cas9 systems have also been applied to C. auris.These systems must be incorporated into the C. auris genome, as to date C. auris has not been shown to be able to maintain plasmids.The permanent incorporation of CRISPR components and selective markers to the genome presents a challenge as these components can limit the ability to further genetically manipulate a strain.Investigators have sought to mitigate this challenge in a variety of ways.Direct transformation of CRISPR Cas9 RNA protein complexes into C. auris using electroporation can be used to edit the C. auris genome [39].Furthermore, recyclable CRISPR-mediated systems that leverage lithium acetate transformation protocols have been optimized for C. auris.Cas9/ sgRNA cassettes marked with either Nat r or hygromycin r also containing regions of homology to the LEU2 locus facilitate insertion of CRISPR components to the genome while simultaneously disrupting LEU2 [40].Cells that have incorporated the CRISPR machinery will grow on drug but require leucine.Once CRISPR-mediated genome editing has been completed, transformants are plated on synthetic complete media lacking leucine to select for the reconstitution of LEU2 by homologous recombination and resultant recycling of Cas9-sgRNA insertions from the genome, thus enabling investigators to serially edit the C. auris genome.

What model organisms can we use to study C. auris pathogenesis?
Animal models that accurately recapitulate C. auris infection are critical to understanding pathogenesis.Although there are differences in skin biology between humans and mice [41], mouse models offer numerous advantages when studying virulence.Genetically modified animals and well-characterized immunological tools are available and are widely used to understand skin infection pathogenesis in vivo [18].For example, a recent study by Huang and colleagues utilized knock out mouse models to dissect the contribution of IL-17-mediated innate and adaptive immune response in C. auris skin colonization [18].Furthermore, recent studies compared the virulence potential of C. auris and other Candida species including C. albicans using immunocompetent and immunocompromised mice models of systemic infection [13,42,43].C. auris elicits less immunoinflammatory response than C. albicans, and this differential response correlates with distinctions in cell wall structure [43].C. albicans is a commensal yeast and it is likelyAU : PleasecheckwhetherthechangesmadeinthesentenceC:albicansisaco its surface determinants have coevolved with the human immune system.C. auris is not an obligate commensal, but can colonize different host niches including skin, gut, and internal organs [18,43,44].Comparing differences between the immune response to these pathogens have the potential to provide insights into how virulence mechanisms between these pathogens differ.
Aside from mouse, other model systems such as Zebrafish [45], Galleria mellonella [46], Caenorhabditis elegans [47], and Drosophila melanogaster [48] are utilized to study C. auris colonization and pathogenesis and can provide distinct advantages to mammalian models.Furthermore, ex vivo models such as human and pig skin can be used to conduct C. auris colonization and biofilm studies [41,[49][50][51].Collectively, depending on the type of experiment, invertebrate whole animal, mouse, and ex vivo skin models can be utilized to investigate fungal virulence and host-pathogen interactions.

What are the current decolonization and disinfection strategies used to control C. auris?
Decolonization in patients and environmental decontamination of C. auris is critical to prevent and control the spread of C. auris infections.Currently, there is no effective skin decolonization strategy for C. auris.Chlorhexidine gluconate (CHG) skin wash is used in some facilities to reduce the skin burden of multidrug-resistant microorganisms including C. auris.However, only high concentrations of CHG (�625 μg/ml), which are rarely achieved on the skin, were shown to be effective for reducing the burden of C. auris on the skin in 1 study [3].Persistent colonization has been reported in spite of using this antiseptic [3,52,53].Further, using effective disinfectants is critical for the prevention and control of C. auris in the environment.The most current CDC recommendations for infection control can be found on the CDC website and include a list of recommended environmental disinfectants and other recommended practices [54].These recommendations for disinfectants were based on recent studies showing efficacy of hydrogen peroxide or alcohol-based active compounds against C. auris and ineffectiveness of products based on quaternary ammonium chemistry (QAC) [55,56].In addition, recently C. auris was also detected in the wastewater samples in Nevada, United States of America [25].Further studies are needed to develop novel strategies to evaluate the efficacy of currently available disinfectants and control measures in real-world settings.